Studies on parabiotic rats and on liver transplantation have suggested that following partial hepatectomy a humoral factor stimulates quiescent hepatocytes to enter the cell cycle. The nature of the serum factors that regulate hepatocyte proliferation has been investigated. A bioassay procedure for the determination of DNA synthesis in primary hepatocyte cultures has been established. This has allowed the purification of a putative hepatocyte growth factor from the serum of hepatectomized rats. In this report we present the following major findings: (1) The partially purified factor isolated from the serum of young Fischer 344 rats produces a dose-dependent stimulation of DNA synthesis in primary hepatocyte cultures. (2) The hepatocyte growth factor appears to be an acid- and heat-labile protein of molecular weight 70-110 KD which has a strong affinity for heparin. (3) The factor also stimulates DNA synthesis in cultures of rat kidney fibroblasts, but is inactive in fetal liver cell cultures and in cultures of hepatoma cell lines. (4) The factor may be a novel growth factor as it has different physical or biological properties compared to known well-characterized growth proteins and polypeptides. (5) There may be a factor(s) present in normal serum which inhibits the activity of the putative hepatocyte growth factor.